title: Two-sided ubiquitin binding of NF-κB essential modulator (NEMO) zinc finger unveiled by a mutation associated with anhidrotic ectodermal dysplasia with immunodeficiency syndrome.
abstract: Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain.
authors.affiliation: Institut Pasteur, Unité de Biochimie Structurale et Cellulaire, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France; Université Pierre et Marie Curie, Cellule Pasteur UPMC, rue du Dr. Roux 75015 Paris, France.
authors.forename: Flora
authors.initials: F
authors.lastname: Ngadjeua
authors.affiliation: Institut Pasteur, Unité de Biochimie Structurale et Cellulaire, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Chiaravalli
authors.affiliation: Institut Pasteur, Unité de Biochimie Structurale et Cellulaire, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France.
authors.forename: François
authors.initials: F
authors.lastname: Traincard
authors.affiliation: Plateforme de Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Raynal
authors.affiliation: Institut Pasteur, Unité de Biochimie Structurale et Cellulaire, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Fontan
authors.affiliation: Institut Pasteur, Unité de Biochimie Structurale et Cellulaire, Department of Structural Biology and Chemistry, CNRS, UMR 3528, 25/28 rue du Dr. Roux 75724 Paris cedex 15, France. Electronic address: fabrice.agou@pasteur.fr.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Agou
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004476:Ectodermal Dysplasia / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007153:Immunologic Deficiency Syndromes / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019169:Jurkat Cells; D051379:Mice; D008817:Mice, Mutant Strains; D008958:Models, Molecular; D020125:Mutation, Missense; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: C491731:IKBKG protein, human; D047908:Intracellular Signaling Peptides and Proteins; C514981:NEMO protein, mouse; D016328:NF-kappa B; D025801:Ubiquitin; D051550:I-kappa B Kinase
keywords: EDA-ID; Fluorescence; IKK Complex; Immunodeficiency; NEMO; NF-κB; Ubiquitin; Zinc Finger